1. Field of the Invention
The present invention relates to an ultrahigh-pressure dual on-line solid phase extraction/capillary reverse-phase liquid chromatography system (hereinafter, abbreviated as a ‘DO-SPE/cRPLC system’ and also referred to simply as a ‘system’. More specifically, the present invention relates to an ultrahigh-pressure DO-SPE/cRPLC system that requires minimal time (i.e. dead time) for column equilibration between successive experiments to shorten the total time required for the experiments by a factor of about two, that enables rapid sample injection, on-line sample desalting and sample enrichment, that is highly reproducible in terms of liquid chromatography (LC) retention time, and that can be operated at a pressure as high as 10,000 psi.
2. Description of the Related Art
On-line solid phase extraction/capillary reverse-phase liquid chromatography is recognized as one of the most powerful analytical tools for current proteomic research due to its high analysis efficiency. On-line solid phase extraction/capillary reverse-phase liquid chromatography has various advantages, and particularly, the ability to effectively separate trace amounts of biological analytes and the wide analyte-solid phase interaction range enable researchers to identify low-abundance proteins with great efficiency.
Mass spectrometry (MS)-based methods for identifications of proteins have become standard analytical platforms in proteomic research. The shotgun approach, or bottom-up approach, which is among the most well-established and robust MS-based strategies, relies on proteolytic digestion of proteins into peptides prior to analysis using a mass spectrometer. Such digestion of proteins increases the solubility of the biological samples and creates peptide fragments that are readily ionized and detected in the mass spectrometer.
This process, however, inevitably results in increased sample complexity. For example, yeast proteome, which—at ca. 6,000 different proteins—is one of the simpler proteomes, will provide over 300,000 peptides. Among the various approaches to overcoming this problem is the use of multidimensional protein identification technology (Link, A. J., Eng, J., Schieltz, D. M., Carmack, E., et al., Nat. Biotechnol. 1999, 17, 676-682; Chen, E. I., Hewel, J., Felding-Habermann, B., Yates, J. R. III, Mol. Cell. Proteomics 2006, 5, 53-56.). However, the efficiency and sensitivity of liquid chromatography (LC) columns remain to be improved significantly. It is already known that the sensitivity of liquid chromatography/mass spectrometry (LC/MS) experiments can be increased dramatically upon decreasing the inner diameters of the separation columns while maintaining their lengths constant (Kim, M.-S., Choie, W.-S., Shin, Y. S., Yu, M. H., Lee, S.-W., Bull. Korean Chem. Soc. 2004, 25, 1833-1839.).
In many cases (e.g., for biological samples that contain substantial amounts of detergents and salts), on-line desalting steps are essential prior to mass analyses because such impurities suppress ionization process of peptide analytes, resulting in decreased detection sensitivity of the analytes. On-line desalting is preferred to off-line methods because the latter are rather time-consuming and also inevitably suffer from sample loss.
Another practical issue when using long, small-inner diameter (ID) capillary columns packed with hydrophobic media is their extended column equilibration (or regeneration) time; in practice, 1-m-long, 75-m-ID columns require at least 2 hours of equilibration before the capillary columns can be reused in subsequent experiments.